Ferrets are widely used for influenza virus pathogenesis, virus transmission, and antiviral therapy studies. However, the contributions of the volume of inoculum administered and the ferrets respiratory tract anatomy to disease outcome have not been explored. We noted variations in clinical disease outcomes and the volume of inoculum administered and investigated these differences by administering two influenza viruses (A/California/07/2009 H1N1 pandemic and A/Minnesota/11/2010 H3N2 variant) to ferrets intranasally at a dose of 106 50% tissue culture infective doses in a range of inoculum volumes (0.2, 0.5, or 1.0 ml) and followed viral replication, clinical disease, and pathology over 6 days. Clinical illness and respiratory tract pathology were the most severe and most consistent when the viruses were administered in a volume of 1.0 ml. Using a modified micro-computed tomography imaging method and examining gross specimens, we found that the right main-stem bronchus was consistently larger in diameter than the left main-stem bronchus, though the latter was longer and straighter. These anatomic features likely influence the distribution of the inoculum in the lower respiratory tract. A 1.0-ml volume of inoculum is optimal for delivery of virus to the lower respiratory tract of ferrets, particularly when evaluation of clinical disease is desired. Furthermore, we identified important anatomical features of the ferret lung that influence the kinetics of viral replication, clinical disease severity, and lung pathology. LID scientists are collaborating with scientists from MedImmune under a CRADA to generate candidate vaccines against avian influenza viruses of each subtype. The vaccines were generated using plasmid based reverse genetics and each contains the hemagglutinin and neuraminidase genes from an avian influenza virus and six internal gene segments from the AA ca virus. H2 influenza viruses have not circulated in humans since 1968 and therefore a significant portion of the population would be susceptible to infection should H2 influenza viruses re-emerge. Therefore the development of an H2N2 vaccine is a priority in pandemic preparedness planning. We previously showed that a single dose of a cold-adapted (ca) H2N2 live attenuated influenza vaccine (LAIV) based on the influenza A/Ann Arbor/6/60 (AA ca) virus was immunogenic and efficacious in mice and ferrets. However, in a Phase I clinical trial, viral replication was restricted and immunogenicity was poor. We compared the replication of four H2N2 LAIV candidate viruses, AA ca, A/Tecumseh/3/67 (TEC67ca), and two variants of A/Japan/305/57 (JAP57 ca) in three non-human primate (NHP) species: African green monkeys (AGM), cynomolgus macaques (CM) and rhesus macaques (RM). One JAP57 ca virus variant had glutamine and glycine at HA amino acid positions 226 and 228 (QG) that binds to alpha 2-3 linked sialic acids, and one had leucine and serine that binds to alpha 2-3 and alpha 2-6 linked residues (LS). The replication of all ca viruses was restricted, with low titers detected in the upper respiratory tract of all NHP species, however replication was detected in significantly more CMs than AGMs. The JAP57 ca QG and TEC67ca viruses replicated in a significantly higher percentage of NHPs than the AA ca virus, with the TEC67ca virus recovered from the greatest percentage of animals. Altering the receptor specificity of the JAP57ca virus from alpha 2-3 to both alpha 2-3 and alpha 2-6 linked sialic acid residues did not significantly increase the number of animals infected or the titer to which the virus replicated. Taken together, our data show that in NHPs the AA ca virus more closely reflects the human experience than mice or ferret studies. CMs and RMs may be the preferred species for evaluating H2N2 LAIV viruses, and the TEC67ca virus may be the most promising H2N2 LAIV candidate for further evaluation. Since it is difficult to predict which influenza virus subtype will cause an influenza pandemic, it is important to prepare influenza virus vaccines against different subtypes and evaluate the safety and immunogenicity of candidate vaccines in preclinical and clinical studies prior to a pandemic. In addition to infecting humans, H3 influenza viruses commonly infect pigs, horses, and avian species. Equine influenza viruses (EIV) are responsible for rapidly spreading outbreaks of respiratory disease in horses. Although natural infections of humans with EIV have not been reported, experimental inoculation of humans with these viruses can lead to a productive infection and elicit a neutralizing antibody response. Moreover, EIV have crossed the species barrier to infect dogs, pigs, and camels and therefore may also pose a threat to humans. Based on serologic cross-reactivity of H3N8 EIV from different lineages, A/equine/Georgia/1/1981 (eq/GA/81) was selected to produce a live attenuated candidate vaccine by reverse genetics with the HA and NA genes of the eq/GA/81 wild-type virus and the six internal protein genes of the A/Ann Arbor/6/60 ca (H2N2) vaccine donor virus. In both mice and ferrets, intranasal administration of a single dose of the eq/GA/81 ca vaccine virus induced neutralizing antibodies and conferred complete protection from homologous wt virus challenge in the upper respiratory tract. One dose of the eq/GA/81 ca vaccine also induced neutralizing antibodies and conferred complete protection in mice and nearly complete protection in ferrets upon heterologous challenge with the H3N8 (eq/Newmarket/03) virus. These data support further evaluation of the eq/GA/81 ca vaccine in humans for use in the event of transmission of an equine H3N8 influenza virus to humans. H3N8 influenza viruses are also commonly found in wild birds, usually causing mild or no disease in infected birds. However, they have crossed the species barrier and have been associated with outbreaks in dogs, pigs, donkeys, and seals and therefore pose a threat to humans. A live attenuated, cold-adapted (ca) H3N8 vaccine virus was generated by reverse genetics using the HA and NA genes from the A/blue-winged teal/Texas/Sg-00079/2007 (H3N8) (tl/TX/079/07) virus and the six internal protein gene segments from the A/Ann Arbor/6/60 ca (H2N2) vaccine donor strain. One dose of the tl/TX/079/07 ca vaccine induced a robust neutralizing antibody response against the homologous (tl/TX/079/07) and two heterologous influenza viruses, including the recently emerged A/harbor seal/New Hampshire/179629/2011 (H3N8) and A/northern pintail/Alaska/44228-129/2006 (H3N8) viruses, and conferred robust protection against the homologous and heterologous influenza viruses. We also analyzed human sera against the tl/TX/079/07 H3N8 avian influenza virus and observed low but detectable antibody reactivity in elderly subjects, suggesting that older H3N2 influenza viruses confer some cross-reactive antibody. The latter observation was confirmed in a ferret study. The safety, immunogenicity, and efficacy of the tl/TX/079/07 ca vaccine in mice and ferrets support further evaluation of this vaccine in humans for use in the event of transmission of an H3N8 avian influenza virus to humans. The human and ferret serology data suggest that a single dose of the vaccine may be sufficient in older subjects.